Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms

Area of Science:

• Nonlinear optics
• Biomedical imaging
• Microscopy

Background:

• Second-harmonic generation (SHG) is a nonlinear optical effect known since early laser physics.
• SHG microscopy has recently emerged as a viable contrast mechanism for cell and tissue imaging.
• SHIM requires only minor modifications to standard two-photon microscopes.

Purpose of the Study:

• To highlight the potential of SHIM as a nondestructive imaging modality.
• To discuss limitations and potential improvements for SHIM.
• To explore SHIM's application in probing cell physiology and neuronal activity.

Main Methods:

• Utilizing second-harmonic generation (SHG) in a laser-scanning microscope.
• Adapting standard two-photon microscopes for SHIM.
• Investigating transmitted light and epi-illumination configurations for SHG signal collection.

Main Results:

• SHIM enables visualization of three-dimensional in vivo structures of protein assemblies like collagen and microtubules.
• Transmitted light geometry currently limits in vivo measurements in large animals.
• SHG signals from membrane dyes show sensitivity to membrane potential.

Conclusions:

• SHIM is a promising nondestructive imaging modality for basic research and clinical pathology.
• Improvements in epi-illumination and dye sensitivity are needed for in vivo applications and imaging fast neuronal activity.
• SHIM holds potential for probing cell physiology and visualizing neuronal electrical activity.